Emerging evidence suggests that gut microbes are pivotal in integrating environmental cues with host physiology and metabolism to influence many chronic conditions, including bone metabolism. This project will extend the provocative findings in animals that the gut microbiome influences the skeleton by conducting a study in humans to test the central hypothesis that the gut microbiome is associated with BMD, microarchitecture and strength. This project is a collaboration between the Framingham Osteoporosis Study and the Osteoporotic Fractures in Men Study (MrOS) that have gut microbiome specimens, high resolution peripheral quantitative computed tomography (HR-pQCT) scans, and essential dietary and lifestyle data to pursue three specific aims that are based on newly generated preliminary data showing an association between gut bacterial taxa and bone microarchitecture. Aim 1 will determine the association between 16S rRNA taxonomic profiles of the gut microbiome and five measures of bone density, architecture and strength measured using HR-pQCT. The study sample (n=3,793) is derived from the Framingham 3rd Generation and Omni Cohorts, and the MrOS Cohort. In aim 2, 864 study participants from the cohorts at the extremes of HR-pQCT derived bone measures, will have whole metagenomic shotgun sequencing performed to refine the associations observed between bone microarchitecture measures, diet, and microbiome taxonomies, and to interrogate the microbiome metabolic potential in relation to bone metabolism using newly developed analytic tools. Finally, in Aim 3, we will measure markers of inflammation, and associate them with microbiome data and BMD, bone microarchitecture and strength to assess whether they are mediating the association between the microbiome and bone. The project is significant because it focuses on osteoporosis, a disease of major public health importance that could benefit from an understanding of the potential effects of the gut microbiome on skeletal health, similar to what has been found for obesity and diabetes. This will be the first large population-based study to use state-of-the-art 16S taxonomic profiling of the gut microbiome and relate this to the most sensitive imaging modality for the human skeleton by a highly qualified team of scientists. The use of whole metagenomic shotgun sequencing in a subset of subjects from the two cohorts is an innovative way to refine the associations observed between bone microarchitecture measures and taxonomies, and to interrogate the microbiome metabolic potential and diet in relation to bone metabolism along with the accompanying inflammatory markers. Overall, the results of this study will provide the best available data on the effects of the gut microbiome on the skeleton, which could lead to interventions targeting the microbiome as a way of improving skeletal health.